Researchers at the Institute for Agrobiotechnology (a mixed research centre set up by the Public University of Navarre, the CSIC-National Scientific Research Council, and the Government of Navarre) are designing, by means of laser application, nanostructured reliefs on surfaces so that they acquire antibacterial properties and are more resistant to the formation of bacterial biofilms. The authors of the research say that in the preliminary tests carried out so far with the bacteria Staphylococcus aureus a reduction in the region of 65-70% has been confirmed in the adhesion of bacteria.

Apart from selecting the materials that best inhibit the adhesion of bacteria, the research is also looking into other aspects. These include the resistance to disinfectants of the bacteria adhered to nanostructured surfaces, how these surfaces retain their properties during prolonged use, or the behaviour of the bacteria on the surface of biomaterials. Topographical patterns that encourage the adhesion of bacteria will also be identified.

The authors anticipate that the applications coming out of this research will have an impact on a broad field from surgical material treated in advance using laser (prostheses, catheters) to water or aquaculture tanks with surfaces that prevent the adhesion of bacteria.

Preventing the creation of a bacterial biofilm

Bacterial biofilm is created when bacteria grow adhered to a surface and are surrounded by a matrix that they themselves produce and which makes them more resistant. "Bacteria," according to the head researcher Jaione Valle-Turrillas, "stick to any surface; it can be the skin, internal organs, surfaces of materials, etc. and they produce this biofilm, a kind of film that makes them more resistant to antibiotic treatments, disinfectants, etc." Biofilms can be found in nature (bacteria adhered to the surfaces of stones in rivers), in our own bodies (intestinal and buccal flora), in filters and pipes, in water tanks, on farms (milking equipment) and in the clinical ambit (prostheses, surgical catheters), etc.

The Biofilms Microbianos research group of the Institute of Agrobiotechnology is working mainly with two bacteria: S. aureus and Salmonella. Various lines of research focusing on the prevention or elimination of biofilms and ranging from the development of vaccines to research into biofilm dispersants, are being pursued in the laboratory, and right now, research is being done in this project to modify surfaces to prevent the formation of biofilm.

"Thanks to DLIP [Direct Laser Interference Patterning] technology, a surface is interfered with and modified using different laser beams on a nanometric scale," explains Jaione Valle. "You can create different patterns and drawings, of different periodicity, from nanometres to micra. We've already tested different surfaces and have found a material and a pattern that will stop the bacteria from sticking to the surface; it does not eliminate them completely, but the reduction is between 65 and 70%."

First of all, the surface is modified by means of laser and then the bacteria are applied to see how they produce the biofilm and in what quantity. Various materials have been used during the tests, and it has been seen how the number of bacteria and the production of biofilm diminish according to bacteria type and type of structure applied to the surface.

To quantify the reduction in the number of bacteria and the extent to which they remain adhered to the nanostructured surface, the researchers used a reagent (Alamar Blue), which emits fluorescence when it comes into contact with live bacteria. "This reaction is measured in a fluorometer so that the more bacteria there are, the greater the fluorescence that is produced," points out the researcher. The problem is that this technique cannot differentiate when the adhesion differences are small. That is why we are now using another method: we collect all the bacteria that have stuck to the surface, we sow them in a culture medium and count the number of colonies; it's more laborious, but it's also much more reliable."

The project "Development and evaluation of antibacterial properties of surfaces with nanostructured reliefs generated by Direct Laser Interference Patterning (DLIP)" is scheduled to run for three years, and will be brought to a conclusion at the end of December 2013. It is being run in collaboration with the German R+D centre Institut Fraunhofer for Material and Beam Technology, which has provided the laser technology to generate the reliefs on the surfaces. The IdAB-Agrobiotechnology Institute, for its part, is conducting the study and experimental tests. The total budget, funded by the Department of Innovation, Companies and Employment of the Government of Navarre, amounts to 179,800 euros.

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